This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 8-Oxoguanine glycosylase 1 (Ogg1) is a constitutive glycosylase responsible for the removal of the promutagenic modified base 8-hydroxy-2'-deoxyguanosine (oxo8dG) from DNA. Ogg1 is expressed in all tissues and it is suggested that its expression is modulated by the redox status of the cell, as evidenced by the presence of antioxidant responsible elements (ARE) in the promoter region. Additionally, reports of altered Ogg1 activity and expression under conditions that shift the redox status support this idea. However, little is known of the specific triggers in the redox milieu responsible for the regulation of its expression or activity. Accumulation of oxo8dG has been linked to neuronal cell loss in major neurological pathologies such as Parkinson's and Alzheimer disease, as well as in amyotrophic lateral sclerosis, and during normal aging. It has been shown that some of these high oxo8dG levels are a consequence of a decreased Ogg1 activity. Although oxo8dG can form and accumulate in DNA due to diverse noxious stimuli, its removal from DNA is solely dependent in the activity of Ogg1. Thus, understanding the cellular mechanisms responsible for the regulation and expression of Ogg1 in neurons is needed to identify pathological and/or toxicological conditions responsible for the accumulation of oxo8dG and the onset of disease. Our data, not only supports the notion that the redox status of the cell plays a major role in Ogg1 regulation, but suggests that the level of reduced glutathione (GSH) is the major component in such regulation. We hypothesize that GSH acts as a molecular switch that regulates the oxo8dG levels in DNA via modulation of Ogg1 expression and/or activity. Additionally, alterations in cell response capacity to repair DNA in distinct cell populations will allow identifying differential neuronal vulnerability. This hypothesis will be tested with the following specific aims. Specific Aim 1. To fully delineate temporal GSH-mediated regulation of Ogg1 expression and its consequences in changes in ox8dG levels in neurons. Neuronal cell lines expressing a pAM/HOGG1promoter-hrGFP vector will be exposed to GSH depleting (L-buthionine-(S,R)-sulfoximine (BSO), Diethylmaleate(DEM)) or GSH inducing agents (N-acetyl cysteine(NAC)), and glutathione ethyl ester (GSHEt);time-course changes in Ogg1 expression will be visualized via changes in GFP expression with a fluorescent cell sorter. Changes in Ogg1 at critical times and/or doses will be further corroborated by Real-Time PCR and Ogg1 activity. Additionally, oxo8dG (HPLC-EC) levels will be assessed in relation to alterations in Ogg1 expression. Specific Aim 2. To determine the degree of neuronal vulnerability associated with Ogg1 expression changes after redox modulation. Neuronal cell lines will be treated with BSO, DEM, NAC GSHEt at doses and times that cause maximum change in Ogg1 expression, as identified in Specific Aim1. Then, cells will be challenged with increasing doses of H2O2, a known inducer of oxo8dG or antimycin A, an inhibitor of mitochondrial respiration and inducer of H2O2 and superoxide production. Specific Aim 3. To determine the necessary and/or sufficient role of recognition sites of transcription factors in the promoter region that allow for basal and inducible expression of Ogg1. Site directed mutagenesis deletion of recognition sites of transcription factors (AP4, Nrf2, and two Sp1) located in the 350 base pair up stream to the translation origin for Ogg1 will be done and expression of green fluorescence protein (reporter gene) will be determined via fluorescence activated cell sorter. The relevance for putative recognition sites in Ogg1 promoter will be established for basal and GSH-modulated expression of Ogg1.